| During the past few decades, natural animal silks, mainly from spiders and silkworms, have attracted considerable interest of researchers for their outstanding mechanical properties that are comparable or even superior to these high performance synthetic materials. However, several factors limit the widespread adoption of silkworm and spider silk in commercial sectors. Therefore, regenerated silk proteins, even silk-like proteins synthesized with the same precisely specified amino acid sequences with natural silk proteins, are used to spin silk-like fibers artificially by several different methods. However, most of the artificial silk fibers behave much worse mechanical properties than those of natural silks. In addition, natural silks are formed under mild physiological conditions, including an aqueous medium, ambient temperature, low hydrostatic pressure while extrusion rates and draw down ratios are both low compared with industrial polymers. These conditions are in sharp contrast to the extreme conditions including high temperature, high pressure, toxic solvents and high draw down ratios for extruding industrial polymers. All these maybe draw to a conclusion that: the unique spinning process of natural fibers is crucial to their mechanical properties. In order to develop biomimetic spinning for the production of tough artificial silk fibers, it is very necessary and important to define the natural spinning conditions and understand the spinning mechanism in the formation process of natural silks, as well as the relationship between this unique spinning process and superior mechanical properties of natural fibers.Studies have revealed that the spinning process in vivo of spider or silkworm is a similarly liquid-crystalline spinning process, accompanied by a conformation changeof silk protein from random coil and/or helical conformation into β-sheet under sheer force and/or extensional flow. In addition, some studies suggest that some other factors also involve in the spinning process effectively. Here, we report the use of Proton Induced X-ray Emission (PIXE), Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) and Atomic Adsorption Spectroscopy (AAS) to investigate the concentrations of six metal elements (Na, K, Mg, Ca, Cu and Zn) at different stages in the silk secretory pathway in the Bombyx mori silkworm. The results showed that the contents of each of these elements increased from posterior part to anterior part of silk gland with the exception of Ca(II) which dropped dramatically in the anterior part.In order to investigate the roles of these six metallic ions played in the natural spinning process, we invented a new method to prepare a concentrated regenerated silk fibroin (SF) solution at the concentration similar to the natural dope. Raman spectra was employed to monitor the effects of these ions at different concentrations on the conformations of such a concentrated SF solution with the help of deconvolution of amide I band in Raman spectra. We also carried out a similar qualitative examination of the effects of these ions on natural SF dope prepared directly from the silk gland. The results showed that different metallic ions have their own effects on the conformation transition of SF, and such effects related to their content in different parts of the silk gland. In general, Mg(II), Cu(II) and Zn(II) were favorable to β-sheet formation and their contents increased from MP to MA part in silk gland. Specially, the small Cu(II) content (1.6×10-3-2) could induce the remarkable conformation transition of SF. K(I) could break down the stable gel network of natural SF while Ca(II) helped to maintain it. This may suggest that the increase in Na(I) and K(I) content from MP to MA part of silk gland and the decrease in Ca(II) content both serve to weaken the stable gel and prepare natural SF macromolecules for β-sheet formation in the distal part of the silkworm's anterior division. These findings may have important implications for our understanding of natural spinning process and may assist in the ultimate development of artificialmethods for spinning strong fibers from silk-like proteins.To illustrate Cu(II) effects on SF more detailedly, here we used a novel method to study the interaction of Cu(II) and SF macromolecules. The method was based on the principle of a kinetic catalytic reaction in which Cu(II) act as a catalyzer to catalyze the fading reaction of neutral red, and from UV spectrophotometric analysis, a very small difference of Cu(II) content in reaction system could be determined. The results showed after the addition of SF into the catalytic system, the catalysis ability of Cu(II) was obviously prohibited that implied the free Cu(II) turned into the coordinated ones by forming a Cu(II)-SF complex. The same result of Cu(II) binding to SF chains was found with X-ray proton spectroscopy (XPS) by detecting the binding energy of Cu 2p from CuCl2 and N 1s, O 1s from SF. Moreover, the results showed that only when Cu(II)/SF<1/2,500 (UV results) or Cu(II)/SF<1/1,000 (XPS results), the SF chains could coordinate Cu(II) effectively. Conclusively, Cu(II) can form a complex with SF to introduce a conformation transition from random coil/helix to β -sheet. Combined the fact that Cu(II) content increased from posterior part to anterior part of silk gland, and continuously increased in the silk fiber in the nature, we have the reason to believe that Cu(II) should be one of the factors in the spinning process (silk-forming process) of Bombyx mori silkworm.Based on the new method of preparing concentrated SF at a concentration similar to the natural dope, here we took wet-spinning method to spin artificial silk protein fibers and compared the properties of these fibers spun either from the different kinds of coagulation bath (salt solutions, alcohol and PEG) or with different treatment techniques (stretch and steam-annealing treatment). As a result, we believed that it was feasible to obtain an artificial silk protein fiber with high performance by wet-spinning the concentrated SF under some controlled conditions, which mechanical properties could be very near, even superior to that of natural silk.
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